The present invention relates to reagents, methods and devices for measurement of analytes and, more particularly, to reagents, methods and devices for the measurement of glucose in the blood.
The monitoring of certain analyte concentrations in the body enables early detection of health risks, and identifies the need for the introduction of therapeutic measures. One of the most commonly monitored analytes is glucose, the blood concentration of which is important in determining the appropriate dosages of insulin for diabetics. Various methods have been developed for monitoring glucose levels in the blood, including the use of electrochemical biosensors. Electrochemical biosensors are based on enzyme-catalyzed chemical reactions involving the analyte of interest. In the case of glucose monitoring, the relevant chemical reaction is the oxidation of glucose to gluconolactone. This oxidation is catalyzed by a variety of enzymes, some of which may contain a bound coenzyme such as nicotinamide adenine dinucleotide (phosphate) (NAD(P)), while others may contain a bound cofactor such as flavin adenine dinucleotide (FAD) or pyrroloquinolinequinone (PQQ).
In biosensor applications, the redox equivalents generated in the course of the oxidation of glucose are transported to the surface of an electrode whereby an electrical signal is generated. The magnitude of the electrical signal is then correlated with concentration of glucose. The transfer of redox equivalents from the site of chemical reaction in the enzyme to the surface of the electrode is accomplished with the use of electron transfer mediators.
A significant problem with the use of electron transfer mediators in biosensors is the instability of these compounds upon exposure to common environmental conditions such as temperature and moisture. As a result, the number of mediators suitable for use in glucose biosensors is quite limited.
U.S. Pat. No. 5,520,786 ('786) to Bloczynski et al. describes families of phenothiazine and phenoxazine compounds suitable for use as electron transfer mediators with the enzymes dihydronicotinamide adenine dinucleotide (NADH), NADPH, and analogs thereof. Cofactor based enzymes such as FAD-glucose oxidase and PQQ-glucose dehydrogenase have several advantages over NAD-based enzymes, including lower cost, higher enzyme activity, increased stability, and bound as opposed to readily dissociable cofactor.
Electron transfer mediators previously used with FAD-glucose oxidase and PQQ-glucose dehydrogenase include quinones, phenzine methosulfate, dichlorophenolindophenol and ferricyanide. Unfortunately, these compounds have proven to be highly susceptible to the environmental agents described above, and result in biosensor reagents of low stability. Thus, mediators are needed which exhibit good stability upon exposure to commonly-encountered environmental agents, and which can be used in flavoprotein- and quinoprotein-based systems.
In addition to the need for biosensor reagents that are stable to the environmental agents described above, it would be desirable to provide biosensor reagents that are stable to the radiation conditions commonly employed in lancet sterilization. Reagents stable to such radiation sterilization could be incorporated into highly user-convenient units in which lancet and biosensor are combined.
The present invention is directed to electron transfer mediators for use in flavoprotein- and quinoprotein-based biosensor reagents, which exhibit improved stability to both environmental interferents and to radiation sterilization.